Measuring the amount of chlorophyll fluorescence of the seed envelope is a good method for the assessment of the maturity and quality of seeds. It appeared that simultaneously with the maturation of the seed, the chlorophyll which is present in the seeds is broken down. Therefore, during the maturation process the amount of chlorophyll in the seed envelope decreases. Coincidently the colour changes from green (among others due to the presence of chlorophyll in the immature seeds) to a colour which is depending on the species being investigated. It also appeared that seeds with cracks in the seed envelope showed higher chlorophyll fluorescent signals. Due to these cracks in the seed envelope, the underlying chlorophyll containing tissue (cotyledons, endosperm or embryo) is uncovered. With equipment known and used for sorting seeds on colour, seeds can be sorted in green classes. However, the discrimination of maturity based on colour is not satisfactory: only large differences in maturity can be measured by colour sorting equipment. Therefore, the amount of chlorophyll of the seed envelope is a better basis to discriminate between seeds which differ in maturity.
With the assessment of the amount of chlorophyll fluorescence according to the invention, high certainty can be obtained on the maturity state of the seeds and whether cracks in the seed envelope are present. This makes it possible to sort the seeds with respect to their maturity and on the appearance of cracks in the seed envelope as described in the examples. The border of a class depends on the species and on the seedlot and is calculated on basis of the distribution of the measured fluorescence of a sample taken at random of the particular seedlot. The quality of the seeds in a class depends among others on the choice of the borders of the class. Generally speaking, regarding seeds which after maturation are located in a dry fruit (like seed of cabbage and carrot) the quality of mature seeds is higher compared to less mature or immature seeds. Seeds which maturate in a moist fruit (like seeds of pepper and tomato) have an optimum in their maturity. Immature seeds, but also seeds which are over-mature, have a lower quality compared to seeds of the optimal maturity. Quality being defined as the seed maturation stage, number and size of cracks in the seed envelope, germination percentage, speed of germination, uniformity of germination, vigour, percentage normal seedlings, health and storability. Seeds with an optimal and uniform maturity and without cracks germinate more uniform and give less abnormal seedlings. Seed treatments like priming have a greater effect (faster and more uniform germination) when the seeds have a certain maturity. Moreover, mature seeds have a better storability than less mature or immature seeds. Immature seeds and seeds with cracks are also more sensitive to infection by diseases. Furthermore, a negative health condition during the development of the seed can disturb the maturation process. This will result in unhealthy seeds with a lower degree of maturity than for healthy seeds.
A method to determine the amount of chlorophyll in seeds is known from the article of Tkachuk and Kuzina, in "Chlorophyll analysis of whole rapeseed kernels by near infrared reflectance", Canadian Journal of Plant Science (1982) 62: 875-884. They used a spectrophotometer to point a light beam of known wavelength onto the seed. After reflection the apparatus determines the fractional absorption of the light beam. Preferably the measurement is done in the 400-2400 nm wavelength range. The reflection spectrum is now a measure for the amount of chlorophyll. The amount of chlorophyll is determined with the aim to keep the amount of chlorophyll of the oil of the pressed seeds as low as possible. The main disadvantage of this method lies in the fact that different wavelengths have to be used, preferably 16, in order to obtain a reliable result. This method is not sensitive enough and too complicated to be used in sorting equipment.
In technology several other methods are known to predict the maturity and quality of moist fruit. S. Gunasekaran, M. R. Paulsen and G. C. Shove measured in "Optical Methods for Non-Destructive Quality Evaluation of Agricultural and Biological Materials", Review Paper, Journal of Agricultural Research (1985), 32, 209-241, the amount of chlorophyll in moist fruit to determine the maturity. They used the principle of light absorption at a wavelength of 670 nm. They do not mention the possibility of measuring the amount of chlorophyll of the seeds of a plant.
The method of light absorption is non-destructive with respect to the seeds which are being measured, but not suitable to sort seeds on the basis of the amount of chlorophyll, because of the low sensitivity.
R. M. Smillie, S. E. Hetherinton, R. N. Grantley, R. Chaplin and N. L. Wade measured in "Applications of chlorophyll fluorescence to postharvest physiology and storage of mango and banana fruit and the chilling tolerance of mango cultivars", Asean Food Journal (1987), 3(2), 55-59, the chlorophyll fluorescence with the intention to measure the photosynthetic activity of the fruit. They investigated the changes in chlorophyll fluorescence in the peel of harvested fruit during maturation or exposed to chilling. The speed at which the chlorophyll fluorescence decreased during chilling was used for selecting chilling resistant cultivars. The method of Smillie et al. takes at least 1 hour for the plant material to be dark adapted and then at least over a time period of two seconds the changes in chlorophyll fluorescence to follow, at low light conditions. They give several examples where chlorophyll fluorescence could be used. They do not mention the possibility of measuring the maturity and quality of seeds. They mention the possibility of measuring the 0-level fluorescence, F0, which is measured directly after the excitation light is turned on, but F0 depends on the photosynthesis. They claim that F0 does not have to correlate with the amount of chlorophyll. They also do not mention the possibility of measuring the prompt chlorophyll fluorescence of photosynthetically inactive chlorophyll in dry seeds and the application of sorting seeds with respect to the chlorophyll fluorescence signal on quality and maturity.
European patent application EP A 0 237 363 discloses a fluorescence measuring device designed to overcome the interference of reflectance in the fluorescence signal and not a device to measure the amount of chlorophyll in seeds with the intention to sort seeds. The present invention does not use the method of correcting for the reflectance of the sample according to claim 1 of EP A 0 237 363 but uses an interference filter with a LED-lamp or laser to prevent reflectance signals in the fluorescence signals.
European Patent Application EP-A 0 434 644 discloses a portable instrument designed to measure the ratio of fluorescence at 690 and 730 for the photosynthetic activity (as mentioned in claim 1 and 2) and to measure the Kautsky effect, which also is a response of photosynthetic activity. It does not mention the use of the instrument for measuring the chlorophyll fluorescence of photosynthetically inactive seeds and to sort seeds on their chlorophyll fluorescence signal on maturity and quality. Dry seeds show no photosynthetic activity and therefore no Kautsky effect.
There exists a destructive method to determine the amount of chlorophyll. This method, based on extraction, is internationally recognised as a standard procedure for the assessment of the amount of chlorophyll of rapeseed and is described by J. K. Daun in "Rapeseed--Determination of chlorophyll content--Spectrophotometric method", International Standard Organization, Geneva, (1992) ISO Method 10519. The method is based upon that dry seeds are ground by a mechanical grinder, whereupon the chlorophyll is extracted by a liquid. With a spectrophotometer in the same way as described by Tkachuk et al., but now in transmission, an absorption spectrum, which is characteristic for chlorophyll of the liquid, is made at three different wavelengths, 625, 665 and 705 nm. From this data the amount of chlorophyll can be calculated. The before mentioned method is clearly destructive since the seeds are ground. In the same way as with the article of Tkatchuk et al. also this method is used to determine the amount of chlorophyll to keep it as low as possible in the oil of the pressed seeds.